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Mesenchymal stem cells prevent the rejection of fully allogenic islet grafts by the immunosuppressive activity of matrix metalloproteinase-2 and -9.

Ding Y, Xu D, Feng G, Bushell A, Muschel RJ, Wood KJ - Diabetes (2009)

Bottom Line: Our results demonstrate that matrix metalloproteinases (MMPs) secreted by MSCs, in particular MMP-2 and MMP-9, play an important role in the suppressive activity of MSCs by reducing surface expression of CD25 on responding T-cells.Significantly, these MSC-mediated protective effects were completely reversed by in vivo inhibition of MMP-2 and MMP-9.In addition, we provide a novel insight into the mechanism underlying the suppressive effects of MSCs on T-cell responses to alloantigen.

View Article: PubMed Central - PubMed

Affiliation: Transplantation Research Immunology Group, Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Oxford, UK. yunchuan.ding@nds.ox.ac.uk

ABSTRACT

Objective: Mesenchymal stem cells (MSCs) are known to be capable of suppressing immune responses, but the molecular mechanisms involved and the therapeutic potential of MSCs remain to be clarified.

Research design and methods: We investigated the molecular mechanisms underlying the immunosuppressive effects of MSCs in vitro and in vivo.

Results: Our results demonstrate that matrix metalloproteinases (MMPs) secreted by MSCs, in particular MMP-2 and MMP-9, play an important role in the suppressive activity of MSCs by reducing surface expression of CD25 on responding T-cells. Blocking the activity of MMP-2 and MMP-9 in vitro completely abolished the suppression of T-cell proliferation by MSCs and restored T-cell expression of CD25 as well as responsiveness to interleukin-2. In vivo, administration of MSCs significantly reduced delayed-type hypersensitivity responses to allogeneic antigen and profoundly prolonged the survival of fully allogeneic islet grafts in transplant recipients. Significantly, these MSC-mediated protective effects were completely reversed by in vivo inhibition of MMP-2 and MMP-9.

Conclusions: We demonstrate that MSCs can prevent islet allograft rejection leading to stable, long-term normoglycemia. In addition, we provide a novel insight into the mechanism underlying the suppressive effects of MSCs on T-cell responses to alloantigen.

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Related in: MedlinePlus

Prevention of DTH responses and protection of allogeneic islet grafts by MSCs is dependent on MMP-2 and MMP-9. A: 7 × 106 responder cells (C57BL/6) or 7 × 106 irradiated stimulator cells (CBA.Ca) were injected into the ear pinnae of BALB/c Rag−.−γ−.− mice either alone or together in the presence or absence of 1 ×105 MSCs. DTH response–induced ear swelling was calculated by subtracting the thickness before injection from the thickness after 48 h. Administration of MSCs led to profound reduction of the alloreactive DTH response, which was reversed in the presence of the inhibitor SB-3CT. Data shown are means ± SD of a representative of at least three independent experiments. B: BALB/c Rag−.−γ−.− were rendered diabetic by a single intravenous injection of 200 mg/kg STZ. Mice had an average blood glucose concentration of 20 mmol/l immediately prior islet transplantation. A total of 500 IE pancreatic islets from CB57/B6 mice (H2b) alone or in the presence of 4 × 105 BALB/c MSCs (H2d) were transplanted under the kidney capsules of STZ-induced diabetic BALB/c Rag−.−γ−.−. In some experiments, MSCs were pretreated with 6 μmol/l SB-3CT for 48 h and mice receiving treated MSCs also received SB-3CT (25 μg/mouse) intraperitoneally once every 4 days from the day of islet transplant until rejection. All islet transplant recipients were reconstituted with 1 × 105 naïve BALB/c CD4+CD25− T-cells as an effector T-cell population. Rejection was defined as a blood glucose >14.5 mmol/l for at least 2 consecutive days. Continued function of the islet allografts was confirmed by removal of the islet-bearing kidneys and a return to hyperglycemia. C: Kidneys transplanted with islets grafts were excised at the time of rejection or the end point of this study. Immunofluorescence examination of islet grafts in MSC-treated recipients revealed intensely staining insulin producing islets beneath the kidney capsule, whereas in the MSC-untreated recipients the insulin-positive islet tissue was not detected at the time of rejection. A high-quality digital representation of this figure is available in the online issue.
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Figure 7: Prevention of DTH responses and protection of allogeneic islet grafts by MSCs is dependent on MMP-2 and MMP-9. A: 7 × 106 responder cells (C57BL/6) or 7 × 106 irradiated stimulator cells (CBA.Ca) were injected into the ear pinnae of BALB/c Rag−.−γ−.− mice either alone or together in the presence or absence of 1 ×105 MSCs. DTH response–induced ear swelling was calculated by subtracting the thickness before injection from the thickness after 48 h. Administration of MSCs led to profound reduction of the alloreactive DTH response, which was reversed in the presence of the inhibitor SB-3CT. Data shown are means ± SD of a representative of at least three independent experiments. B: BALB/c Rag−.−γ−.− were rendered diabetic by a single intravenous injection of 200 mg/kg STZ. Mice had an average blood glucose concentration of 20 mmol/l immediately prior islet transplantation. A total of 500 IE pancreatic islets from CB57/B6 mice (H2b) alone or in the presence of 4 × 105 BALB/c MSCs (H2d) were transplanted under the kidney capsules of STZ-induced diabetic BALB/c Rag−.−γ−.−. In some experiments, MSCs were pretreated with 6 μmol/l SB-3CT for 48 h and mice receiving treated MSCs also received SB-3CT (25 μg/mouse) intraperitoneally once every 4 days from the day of islet transplant until rejection. All islet transplant recipients were reconstituted with 1 × 105 naïve BALB/c CD4+CD25− T-cells as an effector T-cell population. Rejection was defined as a blood glucose >14.5 mmol/l for at least 2 consecutive days. Continued function of the islet allografts was confirmed by removal of the islet-bearing kidneys and a return to hyperglycemia. C: Kidneys transplanted with islets grafts were excised at the time of rejection or the end point of this study. Immunofluorescence examination of islet grafts in MSC-treated recipients revealed intensely staining insulin producing islets beneath the kidney capsule, whereas in the MSC-untreated recipients the insulin-positive islet tissue was not detected at the time of rejection. A high-quality digital representation of this figure is available in the online issue.

Mentions: To test the hypothesis that MMPs play a functional role in MSC-mediated suppression in vivo, we used two models of T-cell responses to alloantigens. In the first, we used the trans vivo DTH assay, where ear swelling provides a read-out of T-cell responses (34), and in the second, we used life-sustaining allogeneic islet transplants in diabetic mice (35). In the DTH assay, allogeneic stimulator and responder cells were injected into the ear pinnae of immune-deficient mice in the presence or absence of MSCs and with or without the MMP inhibitor SB-3CT. As shown in Fig. 7A, coinjection of responders and stimulators resulted in a threefold increase in ear swelling compared with that induced by responders only. This DTH response was virtually abolished by coinjection of MSC, thus demonstrating a clear functional effect of these cells in modulating allogeneic responses in vivo (***P = <0.001). Significantly, however, administration of the SB-3CT (6 μmol/l) in the presence of MSCs reversed this effect, resulting in virtually unmodified DTH responses (***P = <0.001).


Mesenchymal stem cells prevent the rejection of fully allogenic islet grafts by the immunosuppressive activity of matrix metalloproteinase-2 and -9.

Ding Y, Xu D, Feng G, Bushell A, Muschel RJ, Wood KJ - Diabetes (2009)

Prevention of DTH responses and protection of allogeneic islet grafts by MSCs is dependent on MMP-2 and MMP-9. A: 7 × 106 responder cells (C57BL/6) or 7 × 106 irradiated stimulator cells (CBA.Ca) were injected into the ear pinnae of BALB/c Rag−.−γ−.− mice either alone or together in the presence or absence of 1 ×105 MSCs. DTH response–induced ear swelling was calculated by subtracting the thickness before injection from the thickness after 48 h. Administration of MSCs led to profound reduction of the alloreactive DTH response, which was reversed in the presence of the inhibitor SB-3CT. Data shown are means ± SD of a representative of at least three independent experiments. B: BALB/c Rag−.−γ−.− were rendered diabetic by a single intravenous injection of 200 mg/kg STZ. Mice had an average blood glucose concentration of 20 mmol/l immediately prior islet transplantation. A total of 500 IE pancreatic islets from CB57/B6 mice (H2b) alone or in the presence of 4 × 105 BALB/c MSCs (H2d) were transplanted under the kidney capsules of STZ-induced diabetic BALB/c Rag−.−γ−.−. In some experiments, MSCs were pretreated with 6 μmol/l SB-3CT for 48 h and mice receiving treated MSCs also received SB-3CT (25 μg/mouse) intraperitoneally once every 4 days from the day of islet transplant until rejection. All islet transplant recipients were reconstituted with 1 × 105 naïve BALB/c CD4+CD25− T-cells as an effector T-cell population. Rejection was defined as a blood glucose >14.5 mmol/l for at least 2 consecutive days. Continued function of the islet allografts was confirmed by removal of the islet-bearing kidneys and a return to hyperglycemia. C: Kidneys transplanted with islets grafts were excised at the time of rejection or the end point of this study. Immunofluorescence examination of islet grafts in MSC-treated recipients revealed intensely staining insulin producing islets beneath the kidney capsule, whereas in the MSC-untreated recipients the insulin-positive islet tissue was not detected at the time of rejection. A high-quality digital representation of this figure is available in the online issue.
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Figure 7: Prevention of DTH responses and protection of allogeneic islet grafts by MSCs is dependent on MMP-2 and MMP-9. A: 7 × 106 responder cells (C57BL/6) or 7 × 106 irradiated stimulator cells (CBA.Ca) were injected into the ear pinnae of BALB/c Rag−.−γ−.− mice either alone or together in the presence or absence of 1 ×105 MSCs. DTH response–induced ear swelling was calculated by subtracting the thickness before injection from the thickness after 48 h. Administration of MSCs led to profound reduction of the alloreactive DTH response, which was reversed in the presence of the inhibitor SB-3CT. Data shown are means ± SD of a representative of at least three independent experiments. B: BALB/c Rag−.−γ−.− were rendered diabetic by a single intravenous injection of 200 mg/kg STZ. Mice had an average blood glucose concentration of 20 mmol/l immediately prior islet transplantation. A total of 500 IE pancreatic islets from CB57/B6 mice (H2b) alone or in the presence of 4 × 105 BALB/c MSCs (H2d) were transplanted under the kidney capsules of STZ-induced diabetic BALB/c Rag−.−γ−.−. In some experiments, MSCs were pretreated with 6 μmol/l SB-3CT for 48 h and mice receiving treated MSCs also received SB-3CT (25 μg/mouse) intraperitoneally once every 4 days from the day of islet transplant until rejection. All islet transplant recipients were reconstituted with 1 × 105 naïve BALB/c CD4+CD25− T-cells as an effector T-cell population. Rejection was defined as a blood glucose >14.5 mmol/l for at least 2 consecutive days. Continued function of the islet allografts was confirmed by removal of the islet-bearing kidneys and a return to hyperglycemia. C: Kidneys transplanted with islets grafts were excised at the time of rejection or the end point of this study. Immunofluorescence examination of islet grafts in MSC-treated recipients revealed intensely staining insulin producing islets beneath the kidney capsule, whereas in the MSC-untreated recipients the insulin-positive islet tissue was not detected at the time of rejection. A high-quality digital representation of this figure is available in the online issue.
Mentions: To test the hypothesis that MMPs play a functional role in MSC-mediated suppression in vivo, we used two models of T-cell responses to alloantigens. In the first, we used the trans vivo DTH assay, where ear swelling provides a read-out of T-cell responses (34), and in the second, we used life-sustaining allogeneic islet transplants in diabetic mice (35). In the DTH assay, allogeneic stimulator and responder cells were injected into the ear pinnae of immune-deficient mice in the presence or absence of MSCs and with or without the MMP inhibitor SB-3CT. As shown in Fig. 7A, coinjection of responders and stimulators resulted in a threefold increase in ear swelling compared with that induced by responders only. This DTH response was virtually abolished by coinjection of MSC, thus demonstrating a clear functional effect of these cells in modulating allogeneic responses in vivo (***P = <0.001). Significantly, however, administration of the SB-3CT (6 μmol/l) in the presence of MSCs reversed this effect, resulting in virtually unmodified DTH responses (***P = <0.001).

Bottom Line: Our results demonstrate that matrix metalloproteinases (MMPs) secreted by MSCs, in particular MMP-2 and MMP-9, play an important role in the suppressive activity of MSCs by reducing surface expression of CD25 on responding T-cells.Significantly, these MSC-mediated protective effects were completely reversed by in vivo inhibition of MMP-2 and MMP-9.In addition, we provide a novel insight into the mechanism underlying the suppressive effects of MSCs on T-cell responses to alloantigen.

View Article: PubMed Central - PubMed

Affiliation: Transplantation Research Immunology Group, Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Oxford, UK. yunchuan.ding@nds.ox.ac.uk

ABSTRACT

Objective: Mesenchymal stem cells (MSCs) are known to be capable of suppressing immune responses, but the molecular mechanisms involved and the therapeutic potential of MSCs remain to be clarified.

Research design and methods: We investigated the molecular mechanisms underlying the immunosuppressive effects of MSCs in vitro and in vivo.

Results: Our results demonstrate that matrix metalloproteinases (MMPs) secreted by MSCs, in particular MMP-2 and MMP-9, play an important role in the suppressive activity of MSCs by reducing surface expression of CD25 on responding T-cells. Blocking the activity of MMP-2 and MMP-9 in vitro completely abolished the suppression of T-cell proliferation by MSCs and restored T-cell expression of CD25 as well as responsiveness to interleukin-2. In vivo, administration of MSCs significantly reduced delayed-type hypersensitivity responses to allogeneic antigen and profoundly prolonged the survival of fully allogeneic islet grafts in transplant recipients. Significantly, these MSC-mediated protective effects were completely reversed by in vivo inhibition of MMP-2 and MMP-9.

Conclusions: We demonstrate that MSCs can prevent islet allograft rejection leading to stable, long-term normoglycemia. In addition, we provide a novel insight into the mechanism underlying the suppressive effects of MSCs on T-cell responses to alloantigen.

Show MeSH
Related in: MedlinePlus